**2. Tissue culture based approaches for sugarcane crop improvement**

Since the pioneer work on callus induction at Hawaiian Sugar Planters' Association Experiment Station and the method developed by Nickell [11] for root production, sugarcane tissue culture appeared as a valuable tool for diverse research activities. Shortly after this, Heinz and Mee [12] published the first report on sugarcane regeneration. These *in vitro* techniques had huge impact on basic research and also on the research of commercial interest which includes maintenance of elite germplasm, production of agronomically superior somaclones, micropropagation of elite clones, healthy planting material and screening for abiotic/biotic stress tolerance.

### **2.1. Somatic embryogenesis**

sustainably. Various promising varieties have been developed so far but they are posed to certain drastic stresses including biotic as well as abiotic stresses. Similarly, efforts have been made to improve sugar recovery. Since, crop productivity and quality can only be improved by employing innovative technologies. Plant tissue culture and genetic engineering has great potential to resolve problems faced by this crop [2]. Transgenic technology can do a lot to address all the aforementioned yield limiting constraints as any of the alien genes may be introduced into the plant through genetic transformation methodologies. Different methods of transformation i.e. biolistic [3, 4] *Agrobacterium* [5] and electroporation [6] have been employed to engineer valuable agronomic traits like resistance against weedicides [7], viruses [8] and insects [3]. Efforts have been made to engineer metabolic pathways for improved sugar content [9] and for the production of biopolymers and bioplastics. Omics approaches have contributed a lot to understand and explore sugarcane genome to develop improved clones. Milestones in structural and functional genomics are also convincing. Different types of markers have been developed to speed up molecular breeding through early identification of superior genotypes [10]. Thus, biotechnological interventions have great potential to promote sugarcane not only as future energy crop

but also a factory house for the production of therapeutics and industrial compounds.

These interventions have been discussed here to focus critical areas of research that can attract researchers for the improvement of this crop. Understanding molecular mechanisms involved

**Figure 1.** Schematic sketch showing role of biotechnological interventions for the improvement of sugarcane crop and

sugar production.

114 Sugarcane - Technology and Research

Somatic embryogenesis may be divided into two phases: induction and expression. During the induction phase, embryogenic competence is acquired by differentiated somatic cells whereas during expression phase, embryogenic cells differentiate into somatic embryos. Komamine et al. [13] evaluated that embryogenic cells did not require any exogenous stimuli in the form of growth hormones or vitamins at induction state. Rather, competent cells require exogenous stimuli at transitional state in very minute quantities. Somatic embryogenesis may either be direct or indirect. Direct somatic embryogenesis involves development of embryo directly on the surface of explant tissues i.e. stem segments, leaf segments, young inflorescence, zygotic embryo, protoplasts and microspores. Indirect somatic embryogenesis involves an intermediary step of callus induction followed by embryogenesis. Different factors have been found to play key role in the acquisition of embryogenic competence. Exogenous growth regulators promote embryogenic competence by affecting cell polarity, pH gradients and by modifying ionic balance all around the cells. Intracellular pH is very crucial for embryogenic competence acquisition. Another critical aspect is the physical isolation of a cell from others. Embryogenic competence acquisition by somatic cells is regulated by the expression of certain genes which involve either upregulation or down regulation of certain functional genes. In addition, physiological, morphological and metabolic variations are also very important for the acquisition of embryogenic competence. Thus, somatic embryogenesis is a great milestone in sugarcane biotechnology [14]. Originally this method was developed as a substitute of meristem culture and regeneration but now it has become an important component of genetic engineering as well. It has well been exploited for the preservation of mutants and transformed material till their approval or field establishment. Various high yielding, early maturing, high sucrose content and smut resistant varieties with good ratooning ability have also been developed through somatic embryogenesis.

*In vitro* propagation of sugarcane through meristematic tissues responded better as compared with other types of plant tissues. Therefore, significant efforts have been made to explore meristematic tissues for mass multiplication. In the beginning of twenty-first century, some reports highlighted direct regeneration of sugarcane genotypes through thin layer culture of cells from immature leaf or inflorescence [24]. They reported to lessen the time span required for *in vitro* propagation. Significant efforts have been made to establish protocols for direct or indirect sugarcane regeneration. Almost each part of the sugarcane plant has been exploited for callus induction but only inflorescence and immature leaves [25] responded better to morphogenic callus. Callus based regeneration gained significance with the prediction that *in-vitro* induced mutations can play some key role in sugarcane improvement [26]. *In vitro* induced variability is beneficial for the development of new varieties but it becomes undesirable when true-to-type plants are required. Only fewer examples have been quoted to highlight useful variations in callus-derived plants. Meristem culture was successfully used to eradicate chlorotic streak disease, sugarcane mosaic virus [27], white leaf disease and ratoon stunting disease. Combination of meristem culture and heat treatment have proved very effective to eradicate pathogens of Fiji disease [28], SCMV [27] and downy mildew. It is an effective method to eliminate most of the bacterial and fungal diseases and is commonly used to eradicate diseases of unknown etiology as well. Disease free planting material of sugarcane obtained from apices culture is now routinely used for the international exchange of this crop. Researchers have explored that plants regenerated from thin cell layer culture can be used to produce disease free sugarcane plants from the infected ones with *Leifsonia xyli*, SCMV and FDV. *In vitro* culture techniques are used in Brazil and USA to produce healthy planting material for commercial applications. Cryotherapy has also appeared as a proficient method to eliminate phytoplasma from the crop plants and has also been used for long term storage

Biotechnological Interventions for the Improvement of Sugarcane Crop and Sugar Production

http://dx.doi.org/10.5772/intechopen.71496

117

Another important application of *in vitro* techniques that attracted researchers is germplasm conservation [30]. *In-vitro* storage of sugarcane germplasm had been established at the Centre de Cooperation Internationale en Recherche Agronomique pour le Development (CIRAD) in France, Sugarcane Breeding Institute in India, and BSES Limited (formerly the Bureau of Sugar Experiment Stations) in Australia. More than 200 hybrid clones of *Saccharum* spp. were preserved at 18°C for 12 months and no phenotypic modifications were observed in the recovered plants. However, with the advancements in tissue culture techniques, *in vitro* preservation became more valuable for the preservation of genetic resources especially of sugarcane [31]. The minimal medium used in *in vitro* preservation has been used successfully during short and medium term preservation, especially for meristems and shoot apices. Decline in explants metabolic activity is usually achieved by changing physical environment or composition of the media used [32]. The commonly used approaches are: lowering of incubation temperature and use of osmotically active compounds such as sorbitol, mannitol and sucrose. Moreover, growth inhibitors like abscisic acid (ABA) is also frequently used. Various factors i.e. vitamins, salts, osmotic stress and others have been explored by different researchers. Survival rate varied in all these experiments but nobody exploited genetic or cytological

of germplasm or production of disease free plants [29].

**2.4. Germplasm conservation**
